There has been a dramatic increase in the need for the chemical analysis of food and agricultural products in recent years. This comes as a result of many factors, some which include increased use of pesticides and fungicides (especially in the developing world), increased regulation and taxation by local and federal governments as well as increased concern about contamination and adulteration of food products. Pest control in intensive agriculture involves treatment of crops (fruits, vegetables, cereals, etc.) pre- and post-harvests with a variety of synthetic chemicals generically known as pesticides. The resurgence of ‘organic’ foods in the last decade has spurred a closer examination of the pesticide and herbicide content of foods consumed. ‘Organic’ is a labeling term that refers to agricultural products produced in accordance with Organic Foods Production Act and the National Organic Program (NOP) Regulations. The principal guidelines for organic production are to use materials and practices that enhance the ecological balance of natural systems and that integrate the parts of the farming system into an ecological whole. Organic agriculture practices cannot ensure that products are completely free of residues; however, methods are used to minimize pollution from air, soil and water.
Herbicides and insecticides are mainly used in the pre-harvest stages, rodenticides are employed in the post-harvest storage stages, and fungicides are applied at any stage of the process depending on the crop. These chemicals can be transferred from plants to animals via the food chain. For example, more than 800 different kinds of pesticides are used for the control of insects, rodents, fungi and unwanted plants in the process of agricultural production. Although most of these are meant to degrade in soil, water and atmosphere before the food product reaches the consumer's table, trace amounts of these pesticide residues can be transferred to humans via the food chain, being potentially harmful to human health [1].
To limit the acceptable risk levels of pesticide residues, federal regulations on maximum residue limits (MRLs) for pesticide residues in foods have been established in many countries and health organizations, for example in the United States, Japan, European Union, and Food and Agriculture Organization (FAO). They are set for a wide range of food commodities of plant and animal origin, and they usually apply to the product as placed on the market. MRLs are not simply set as toxicological threshold levels, they are derived after a comprehensive assessment of the properties of the active substance and the residue behavior on treated crops. These legislative limits have become stricter than ever due to the concerns of food safety and the demands of trade barriers, driving the demand for more sensitive and reliable chemical analysis methods for pesticide residues [2].
The chemical analysis of these residues in foods currently requires both extensive sample preparation and expensive analytical instrumentation. Most pesticide residue detection methods for food samples comprise two key preparation steps prior to identification/quantification: extraction of target analytes from the bulk of the matrix, and partitioning of the residues in an immiscible solvent and/or clean-up of analytes from matrix co-extractives, especially fat which interferes with assays. Although there has been significant advancement in the sophistication and power of analytical instruments [3], the ultimate detection limits and quantification accuracy are still primarily influenced by interferences from food matrices [4] [5] [6] [7]. Thus, sample preparation is the bottleneck for the effective and accurate chemical analysis of trace pesticide residues [4] [5].
The aim of sample preparation is to isolate the trace amounts of analytes from a large quantity of complex matrices and eliminate the interferences from the food matrix as much as possible. Typical sample preparation steps include the sampling/homogenization, extraction, and clean-up. Among them, the extraction and clean-up steps play a critical role in the success of pesticide residue chemical analysis. The traditional sample extraction methods, especially liquid-liquid extraction (LLE), have been widely used for pesticide residue chemical analysis.
However, most of these methods are time consuming and use large quantities of organic solvents to remove interference. Recent analytical developments have attempted to minimize the number of physical and chemical manipulations, the solvent volumes, the number of solvent evaporation steps, the use of toxic solvent, and have aimed to automate the extraction and clean-up procedures as far as possible. These include: supercritical-fluid extraction (SFE), pressurized-liquid extraction (PLE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), gel permeation chromatography (GPC), solid-phase extraction (SPE), molecularly imprinted polymers (MIPs), matrix solid-phase dispersion (MSPD), solid-phase micro-extraction (SPME), QuEChERS, cloud point extraction (CPE) and liquid phase micro-extraction (LPME).
Analysis of Naturally Occurring Molecular Components of Agricultural Products
Another area of interest is the chemical analysis of intrinsic molecular components in food products that are regulated for economic or health reasons. Examples include alcohol in beer, liquor or spirits, caffeine in coffee, nicotine in tobacco products and cannabinoids in marijuana-based products. Rather than address all of these products, we will consider, as an example, the regulation of cannabinoids in various products. Numerous methods for identifying Cannabis constituents have appeared in the literature dating back to 1964 [8]. Some of these techniques were very simple, involving TLC on silica gel plates with visual detection by color reaction [9] [10] [11] [12] [13] [14]. The development of hyphenated chromatographic techniques has enabled positive identification of the major components of Cannabis samples. These techniques include gas chromatography with mass spectrometry, diode-array ultraviolet absorption detectors (DAD) in conjunction with high-performance liquid chromatography (HPLC), and UV/Visible wavelength scanners in conjunction with thin-layer chromatography (TLC). These techniques allow identification of the three main neutral Cannabis constituents (FIG. 1)—cannabidiol (CBD), Δ-9-tetrahydro-cannabinol (Δ9-THC) and cannabinol (CBN)—by comparison with published data in each area. HPLC using normal or reversed phases and detection by absorption at different wavelengths [15] [16] [17] [18] [19] [20] or electrochemical means [21], and more complex techniques combining capillary or packed-column GC with mass spectrometry [22] [23] [24] [25] [26].
Gas chromatography coupled with mass spectrometry (GCMS), seems to have emerged as the method of choice for chemical analysis of cannabinoids in hemp food products [22] [23] [24] [25] [26]. The official method of the European Community for the quantitative determination of THC in hemp varieties [27] uses gas chromatography with a flame ionization detector. On the basis of THC content Cannabis plants are divided into fiber-type and drug-type plants. The ratio (THC+CBN)/CBD has been proposed for distinguishing between the phenotypes of Cannabis plants; if the ratio obtained is greater than 1, the Cannabis plant is classified as drug-type; if it is less than 1, it is a fiber-type.
After the legalization of fiber-hemp cultivation in many parts of the world, hemp food products, mostly sold in esoteric stores, were eaten, because of supposed psychoactive properties associated with a potential THC content. Positive drug tests for marijuana use have been reported after ingestion of hempseed oil and other hemp foods. Since the mid 1990's, hemp food has gradually expanded into the natural product market and is increasingly found in natural food stores sold for nutritional and health benefits. A wide variety of hemp-based products is available, including hemp leaves (tea), hemp seed and seed derivatives, oil, flour, beverages (beer, lemonade), and cosmetic products. Hemp food products, even from fiber-type Cannabis varieties, generally contain measurable amounts of THC. Previous analyses of hemp seed oil have revealed a wide range of THC concentrations between 11.5-117.5 mg kg-1 and 7-150 mg kg-1. For sample preparation, all these methods use traditional liquid-liquid extraction (LLE), which is time-consuming and requires large volumes of solvents.
Sample Preparation
For “dirty” samples, e.g., plant materials, GC used with vaporizing injection techniques is most suitable. “Classical” hot split-less injection of a solvent extract of the plant material is the most frequently applied injection technique, however, some adverse effects such as discrimination of low volatiles, sorption and thermal degradation can occur. Another alternative to classical hot split-less injection is programmable temperature vaporization (PTV). This injection technique, first introduced in 1979, comprises injection of the sample into the cold liner (temperature held below or near the solvent boiling point) and subsequent increase of temperature and transfer of analytes. This technique was shown to avoid discrimination of low volatile compounds and avoid degradation of thermally unstable analytes. The main advantage of PTV, however, includes the possibility of large volume injection (LVI). In the solvent split mode, the PTV allows one to introduce up to 1 ml of sample into the GC system. Injection of large sample volumes not only system. Injection of large sample volumes not only enables significant improvement of overall sensitivity of the analytical method, but also makes the PTV injector applicable for the on-line coupling of GC techniques with various clean-up and enrichment techniques. Otherwise, most analytical procedures require extensive extraction and concentration enhancement steps that make the chemical analysis fairly complex.
Typical procedures used to extract neutral cannabinoids utilize solvent extraction of the plant material. The extracts are obtained by ultrasound mixing (for 15 minutes) of each of the samples, in the ratio of 100 mg of substance to 10 ml of solvent (a mixture consisting of 90 percent hexane and 10 percent chloroform), after which the extracts are ultra-centrifuged for 15 minutes at 10,000 revolutions per minute to isolate the clear supernatant. Solid-phase microextraction (SPME), discovered and developed by Pawliszyn and co-workers [28], has recently emerged as a versatile solvent-free alternative to these conventional liquid-liquid extraction procedures.
Headspace solid-phase microextraction (HS-SPME) is based on the distribution of analytes between the sample, the headspace above the sample, and a coated fused-silica fiber. Analytes are absorbed by the coating of the fiber, where they are focused, until the concentrations in the phases are in equilibrium. Subsequently, the fiber can be injected directly into a GC injection port for thermal desorption. Headspace extraction contrasts with extraction of the analytes by dipping the fiber into the aqueous phase (direct immersion, DISPME) and is advantageous because the low matrix interferences result in a diminished chromatographic background, solvent consumption is markedly reduced and its overall technical performance is fast and simple. The use of SPME in food chemical analysis was recently reviewed by Kataoka [29].
A more complete approach for the chemical analysis of all cannabinoids in plant samples uses heat to induce the decarboxylation of acidic components. Typically, neutral cannabinoids are formed during storage of the plant material but, in order to obtain total cannabinoid in the neutral form, Smith [30] heated the plant material at 100° C. for 6 min under a nitrogen purge. Later investigations showed that stronger heating for prolonged times (i.e. 200° C. for 30 min) caused loss of neutral cannabinoids by evaporation even when the samples were treated in screw cap culture tubes under an atmosphere of nitrogen [31]. Heating plant material at 37 and 60° C. gave significantly different results for neutral cannabinoids [32].
Veress et al. [33] investigated decarboxylation of cannabinoid acids in an open reactor in a study which involved different solvents (n-hexane, ethylene glycol, diethylene glycol, n-octanol, dioctyl phthalate and dimethylsulphoxide), different temperatures and heating times, and various decarboxylation media, for example glass and various sorbent surfaces. The conclusion was that the optimum conditions for the decarboxylation of cannabinoid acids, in the presence or absence of organic solvent, always required temperatures at which the neutral cannabinoids evaporated. Consequently, it is not possible to bring about the conversion of cannabinoid acids into equivalent amounts of neutral cannabinoids by simply heating in an open reactor. It appears that the best conditions for the decarboxylation of cannabinoid acids in closed reactors (screw cap culture tubes) involve heating the samples at 200° C. for just 2 min [31].
Sample Handling and Tracking
In many cases, it is difficult to track samples, especially when the sample material is not directly connected to a sub-sample, i.e., the sample extract. In many instances, sample tracking can be facilitated through the use of Automatic Identification and Data Capture (AIDC), a term frequently used to describe the identification of articles and collection of data into a processor controlled device without the use of a keyboard. AIDC technology is designed to increase efficiency in collection and identification by reducing errors and increasing the rate of identification and collection. For the purposes of automatic identification, a product item is commonly identified by a 12-digit Universal Product Code (UPC), encoded machine-readably in the form of a printed bar code. The most common UPC numbering system incorporates a 5-digit manufacturer number and a 5-digit item number. Because of its limited precision, a UPC is used to identify a class of product rather than an individual product item. The Uniform Code Council and EAN International define and administer the UPC and related codes as subsets of the 14-digit Global Trade Item Number (GTIN).
Within supply chain management, there is considerable interest in expanding or replacing the UPC scheme to allow individual product items to be uniquely identified and thereby tracked. Individual item tagging can reduce “shrinkage” due to lost, stolen or spoiled goods, improve the efficiency of demand-driven manufacturing and supply, facilitate the profiling of product usage, and improve the customer experience.
There are two main contenders for individual item tagging: visible two-dimensional bar codes, and radio frequency identification (RFID) tags. Bar code symbols and bar codes represent one type of AIDC technology. Bar codes have become ubiquitous parts of everyday commercial transactions. Merchandise carried by grocery stores, for example, is labeled with a barcode. A scanner is used to identify an item at the point of purchase by the consumer. The scanner uses the bar code information to look up the item's price. The price is then provided to a cash register for tallying the customer's bill.
Bar codes traditionally consist of a sequence of two element types: bars and spaces. The bars and spaces are arranged such that the bars are parallel and the spaces separate the bars. One encoding methodology varies the width and the sequence of the elements to encode alphanumeric data. The particular encoding methodology is referred to as a barcode symbology. An optical scanner is used to read the bar code symbol and decode the bar code to provide the original alphanumeric data.
The use of the data may vary depending upon the needs of the inquiring entity. A grocery store, for example, may need a unique identifier for a particular product in order to enable calculation of price at checkout or for managing inventory. A medical supplier, however, may need to identify manufacturing dates, lot numbers, expiration dates, and other information about the same product to enable better distribution control. The level of identification needed may vary depending upon the intended use.
Bar code symbologies are efficiently designed to support a specific industry need rather than a wide range of needs. A number of bar code symbologies are presently being used to track products throughout their life expectancy as they are manufactured, distributed, stored, sold, serviced, and disposed of. The bar code symbology designed for one application, however, may not suffice the needs of another application.
Bar codes have the advantage of being inexpensive, but require optical line-of-sight for reading and in some cases appropriate orientation of the bar code relative to the sensor. Additionally, they often detract from the appearance of the product label or packaging. Finally, damage to even a relatively minor portion of the bar code can prevent successful detection and interpretation of the bar code.
RFID tags have the advantage of supporting omnidirectional reading, but are comparatively expensive. Additionally, the presence of metal or liquid can seriously interfere with RFID tag performance, undermining the omnidirectional reading advantage. Passive (reader-powered) RFID tags are projected to be priced at 10 cents each in multi-million quantities by the end of 2003, and at 5 cents each soon thereafter, but this still falls short of the sub-one-cent industry target for low-price items such as grocery. The read-only nature of most optical tags has been cited as a disadvantage, since status changes cannot be written to a tag as an item progresses through the supply chain. However, this disadvantage is mitigated by the fact that a read-only tag can refer to information maintained dynamically on a network.
A two-dimension barcode is a new technology of information storage and transmission, which is widely used in various applications, including product identification, security and anti-counterfeiting, and E-commerce. The two-dimension barcode records information data with specific geometric patterns of black and white graphic symbols arranged in two-dimensional directions. The concept of logical basis of “0” and “1” bit stream adopted in computer systems is utilized to form graphic symbols that correspond to binary representation of text and numerical information. The graphic symbols can be read by an image input device or a photoelectric scanning device to achieve automatic information processing.
International standards of the two-dimension barcode include for example PDF417, Data Matrix, Maxi Code, and QR (Quick Response) Code, among which QR code is most widely used. The QR code shows an advantage of high-speed and all-direction (360 degrees) accessibility, and is capable of representation of Chinese characters, rendering QR code wide applicability in various fields. The QR code comprises a square array of a series of small square message blocks, in which “0” or “1” are represented through variation of gray levels of bright and dark blocks.
Chromatographic and Mass Spectrometric Analysis
GC is the most widely used technique in herbicide and cannabinoid chemical analysis, but it cannot be used directly to analyze all cannabinoids owing to limitations in volatility of the compounds. chemical analysis of Cannabis by GC has been reviewed [34]. Although the cannabinoids have very similar structural features, adequate separations of most of these compounds have been achieved on a number of commercially-available stationary phases. The most widely used are fused silica non-polar columns such as HP-1 and HP-5 as well as DB-1 and DB-5. Identification of the constituents is most readily performed by MS: un-derivatized 1, 3 and 6 show characteristic peaks at m/z values of 314, 246, 231, 193, 174 and 121, of 314, 299, 271, 231 and 55, and of 310, 296, 295 and 238, respectively [35].
Although GC chemical analysis is suitable for plant cannabinoids, the method is restricted to the determination of the quality of Cannabis for smoking if used directly since it can only provide information about the decarboxylated cannabinoids such as Δ9-THC [17]. Many GC reports concern non-derivatization methods because the target of most chemical analysis is the main neutral cannabinoids, and also because it is very difficult to obtain a complete derivatization of a sample for the purposes of quantification. The carboxyl group is not very stable and is easily lost as CO2 under influence of heat or light, resulting in the corresponding neutral cannabinoids: THC, cannabidiol (CBD) and cannabigerol (CBG) [36]. These are formed during heating and drying of harvested plant material, or during storage and when Cannabis is smoked [37] [38] [39].
The variable conditions during all stages of growing, harvesting, processing, storage and use also induce the presence of breakdown products of cannabinoids. The most commonly found degradation product in aged Cannabis is cannabinol (CBN), produced by oxidative degradation of THC under the influence of heat and light [40]. In order to quantify the “total THC content” once present in the fresh plant material, the concentrations of degradation products have to be added to THCA and THC contents.
A number of compounds have been used successfully as internal standards for quantitative chemical analysis. In particular, 5α-cholestane (Matsunaga et al., 1990), docosane (Ferioli et al., 2000) and tetracosane (Stefanidou et al., 2000) are commonly employed because of their suitability for use with a flame ionization detection (FID). A recent development involves the use of deuterated cannabinoids as internal standards when MS detection is employed. Hexadeuterated (d6)-Δ9-THC gives a better linearity of measurement than (d3)-Δ9-THC (Joern, 1992) and can also be used as a standard in HPLC because it has a different retention time than 3. Ross et al. (2000) employed (d9)-Δ9-THC as a reference compound in order to demonstrate that no cannabinoids are present in Cannabis seeds even in the drug phenotype: the cannabinoids often found on the seed surface probably arise from contamination during harvesting.
Electrochemical Techniques
Previous work has shown that it is possible to detect the phenol part of complex molecules by reaction with an electrochemically-generated reagent [41]. In this protocol, the loss of dichloro-benzoquinone monoamine can be monitored electrochemically as it reacts with the substituted phenol of choice. Known as the Gibbs reagent (FIG. 2), it has been used to detect substituted phenols spectrophotometrically, where it has been observed that the most easily displaced substitutes (good anionic-leaving groups) give rise to high yields of dichloroindophenol, while methylphenol and longer alkyl group substitutions such as hydroxybiphenyl, ethylphenol and hydroxybenzoic acid gave no detectable colored product. It has been reported that phenol and phenoxyphenol give good yields of colored products (60 and 63%, respectively), methylphenol gives a low yield (18%), while nitrophenol produces a negative Gibbs reaction [42]. However, this technique is based on observing the product of the Gibbs (or related) reaction, not the consumption of the reagent.
A range of substituted phenols were investigated to determine the versatility of the indirect voltammetric method. This technique is based on the electrochemical oxidation of 2,6-dichloro-p-amino-phenol dissolved in aqueous solution which produces quinoneimine (QI) as shown in FIG. 3. On addition of Δ-THC the reduction wave, corresponding to the electrochemical reduction of quinoneimine (QI) back to aminophenol (AP), as shown in FIG. 3, reduces in magnitude since the QI chemically reacts with Δ-THC providing a useful analytical signal. This methodology is extremely attractive since it avoids the direct oxidation of Δ-THC which can lead to electrode passivation [43]. In similar work, graphite powder was modified with 4-amino-2,6-diphenylphenol which was abrasively immobilized onto a basal plane pyrolytic graphite electrode and assessed for the indirect electrochemical sensing of Δ-THC in saliva [44]. In this way, the detection technique based on the electrochemical formation of the QI was entirely surface confined in respect of the specific agent detecting the Cannabis related material.
Immunoassay Techniques
Immunoassays seem promising for studying cannabinoid metabolites because they are very sensitive, they are able to identify a small class of closely related compounds, and they can be applied directly to the sample without prior extraction or purification. The major problem with immunoassays is, however, one of selectivity. These methods need high-affinity, specific antibodies, but obtaining a very specific antibody that will only bind to one specific antigen is not an easy task since most antibodies bind to a group of closely related compounds. Thus, while immunoassays are particularly suited for screening purposes, positive immunoassay tests should be followed by further confirmative chemical analysis to exclude false positive results [45] [46]. Indeed, according to recent European Union recommendations on testing for drug abuse, and to the USA Mandatory Guidelines for Federal Workplace Drug Testing Programs, chromatographic techniques should always be used to confirm the results obtained by screening with immunoassays [46].
Four main immunoassay techniques are used in screening for cannabinoids, namely, radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), and enzyme-linked immunosorbent assay (ELISA). All of these methods are based on the competitive binding of a labeled antigen and unlabeled antigens from the sample with a limited, known amount of an antibody in the reaction mixture. The RIA and FPIA strategies are very similar in that both determine unbound antigen by either radioactive or fluorescent measurement. In RIA, the bound antigen should be separated from the unbound antigen before radioactivity measurement and, for this purpose, a second antibody is required. The principle of FPIA is that the fluorophore on the free antigen will emit light at a different plane compared with that on the bound antigen.
The measurement of the retention of polarization may be performed without physically separating the bound and the unbound antigens [47]. EMIT is based on the absorbance change produced by the reduction of NAD to NADH coupled to the oxidation of glucose-6-phosphate to 6-phosphogluconolactone, a reaction catalyzed by the enzyme glucose-6-phosphate dehydrogenase attached to the free antigen. The concentration of analyte in the sample determines the amount of free antigen that is labeled with the enzyme, and this is indirectly determining the change in absorbance that is measured [47]. Currently there is only one report of the chemical analysis of plant cannabinoids by immunoassay [48] in which Δ9-THC was measured in a methanolic leaf extract by FPIA using a highly selective monoclonal antibody. The result was confirmed by GC and the immunoassay showed good linear correlation (r=0.977) with the chromatographic method.
Drawbacks and Limitations of Previous Approaches
While tremendous advances have been made in many aspects of the process of sampling volatile components of many samples, the analytical process is still largely time-consuming and expensive, requiring sophisticated technology and highly trained individuals to perform the chemical analysis. There is a great need for simpler and less expensive processes to make such analyses available to a wider audience, who have less technical experience and smaller budgets available for analytical work. Examples of situations where such analytical work would really benefit the customer include groceries and food stores, where staff and customers could ascertain the “organic” quality of grains, produce and meats through a rapid chemical analysis of the content of pesticides, herbicide and other potential contaminants of the commodities that they are buying; the growers and distributers of such commodities, such that they could guarantee the “organic” quality of their products; microbreweries and home brewers, who wish to ascertain the quality of the grains, rice and other commodities used in brewing beer; tobacco farmers and distributors, who wish to determine the nicotine content of tobacco leaves and other products during harvest and distribution; medical marijuana growers, dispensaries, regulators and customers, who wish to ascertain the THC content of hemp and marijuana leaves and other products during harvest and distribution, so as to ascertain the value of their commodities and certify the potency of their products. Therefore, there is a great need for a new technology that separates the sampling process from the chemical analysis process, so as to make the overall chemical analysis more widely available to a larger, less technical market.
In the present disclosure, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which the present disclosure is concerned.
While certain aspects of conventional technologies have been discussed to facilitate the present disclosure, no technical aspects are disclaimed and it is contemplated that the claims may encompass one or more of the conventional technical aspects discussed herein.